선택한 단어 수는 입니다.
최소 단어 이상 선택하여야 합니다.
최소 단어 이상 선택하여야 합니다.
선택한 단어 수는 30입니다.
최대 10 단어까지만 선택 가능합니다.
최대 10 단어까지만 선택 가능합니다.
제안 방법
- Because single-PCR detection of each pathogen is too laborious, time consuming and wasteful of reagents, we attempted to develop mPCR for this purpose. The aim of this investigation was to develop an mPCR assay that could be used for the simultaneous detection of Salmonella, Leptospira and Brucella in a single reaction with highly sensitivity and specificity, using as template either DNA extracted directly from tissue samples or bacterial culture. The availability of such a rapid test should simplify and streamline diagnostic testing for these three important pathogens of humans and animals.
- To optimize the mPCR, we tested all of the primers with several combinations of annealing temperature, extension time, number of cycles and primer concentration. The concentration of magnesium chloride was varied from 1 to 3 mM.
- Our recent attempts to use individual PCR for the detection of those three pathogens in various tissues of wild rodents and cats demonstrated that this technique is relatively costly and time-consuming. Therefore, in this study, we improved the diagnostic value of these PCR methods by taking a multiplex approach. The mPCR has the advantage of simultaneous detection of multiple pathogens and has been proven to be sensitive, specific and cost-effective.
- For the genus Salmonella, the invA primer pair is specific [26], but no studies have evaluated the specificity of these primers for all species in the genus. In this study, to further examine the specificity of each primer pair used in mPCR, all 4 strains of Brucella, 3 strains of Leptospira and 4 strains of Salmonella were tested and gave specific amplification of the correct predicted product size. In addition, the specificity of the primers used for mPCR has also been tested against 15 closely related bacterial species and a variety of other common pathogens, and the absence of amplification of the DNA from these bacterial species indicates that this assay is highly specific for Brucella, Leptospira and Salmonella.
- All mPCR reactions were performed in a thermocycler (PC100; MJ Research, USA) with the following temperature cycling parameters: initial denaturation at 95℃ for 5 min followed by 40 cycles of denaturation at 95℃ for 30 sec, primer annealing at various temperatures from 54 to 60℃ for 30, 40, 50 or 60 sec for optimization, primer extension at 72℃ for 30 or 60 sec, and a final extension at 72℃ for 15 min.
대상 데이터
- All reference strains of Salmonella (S. Enteritidis and Typhimurium), Leptospira (L. interrogans serovar Pomona, L. interrogans serovar Icterohemorrhagiae, and L. interrogans serovar Bratislava), Brucella species (B. abortus, B. abortus vaccine strain RB51, B. ovis and B. canis) and another 15 bacterial strains were used in this study (Table 1).
- Three pairs of oligonucleotide primers were designed to amplify specific regions of BCSP31, LipL41 and invA from the Brucella, Leptospira and Salmonella genomes, respectively. Primer BCSP31 was designed to amplify an approximately 223 bp fragment from Brucella.
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